In vitro analysis is carried out in many environments in order to identify biological samples such as microorganisms, cell and tissue cultures, cellular or sub-cellular extracts, and purified molecules. Samples of various materials are isolated from their usual biological context and provided with an environment in which they can grow. This environment is often provided in the form of a Petri dish which is placed into an incubator in order for the samples to grow. The Petri dish normally includes a microbiological culture medium which encourages growth of the sample. Ideally, incubation on an appropriate culture medium gives rise to the growth of a number of colonies of the sample. Subsequent analysis of the colonies is generally carried out to identify the microorganisms and assess their sensitivity or resistance to antimicrobials.
An important part of the analysis of the samples is the ability to identify particular microorganisms or bacteria, for example, in the colonies. In addition, the treatment of bacteria with appropriate medication can also be analyzed based on the growth of the microorganisms in the sample and the interaction with any medication applied to the sample.
Much of the preliminary analysis is carried out by visual analysis of the Petri dish by qualified scientists. Preliminary visual analysis works well, but is prone to human error and inconsistency due to the huge diversity of shapes, colors, sizes and forms of the different microorganisms which may be difficult to interpret. However, visual inspection is still one of the best ways to quickly identify microorganisms at present.
In addition, as much of the growth is “random”, it is not easy to model microorganism growth and find automated systems which lend themselves to the diversity identified above.
Known incubators may include a window through which samples can be viewed, but in general, the Petri dish is taken out of the incubator to be visually analyzed. Preliminary visual analysis involves holding the Petri dish in front of a light source to identify colonies. Further detailed chemical and microscope analysis methods can then be carried out on particular identified colonies, as required.
Biological scanners, i.e. devices used to scan or count bacterial colonies, are known in the state of the art. For example, US 2004/0101951 and US 2010/0232660 both disclose biological scanners for scanning biological growth plates having different structures but both having in common the ability to generate images of the plates and perform an analysis of these images to detect biological growth. However, both use a single light source providing front or back illumination. Indeed, it is stated in both US2004/0101951 and US2010/0232660 that “some biological growth plates may require only front or back illumination, but not both.” Such illumination is basic and does not allow images of a sufficient quality to carry out a preliminary analysis in an efficient manner to be obtained.
Certain prior art systems exist in which a sample in a Petri dish is illuminated by different colors or wavelengths of light in order to form images of the sample. The images are captured by an appropriately orientated camera.
FR2926820 discloses a method for detecting at least one specific microorganism in a biological sample, said method comprising, amongst others, the step of subjecting a culture medium to at least two radiations, each presenting a specific wavelength. Preferably, two lighting systems are used, each lighting system emitting radiation of a specific wavelength. More specifically, FIG. 1 of FR2926820 shows the combination of visible top lighting and ultraviolet backlighting. The subsequent combined image from the two different illuminations is then used to detect the presence of specific microorganisms.
Similarly, published Japanese patent application, JP2010104301, describes, amongst others, a method for detecting microorganisms comprising an imaging step to take an image of a culture medium on which microorganisms grow and a colony detection step, said method also using a combination of top lighting and backlighting.
Once an image of a biological sample has been obtained, processing techniques can be used to enhance the image. However, there are many problems associated with enhancing images of biological samples. These problems may relate to:                the sizes of colonies being viewed;        the proximity of one colony to another;        the color mix of the colonies;        the nature of the Petri dish;        the nature of the culture medium        etc.        
A first problem of the present invention relates to the determination of the neighborhood of a colony to provide guidance regarding the picking process of the colony. It appears that there is a need to determine one or more isolation areas around a colony.
Another problem relates to the presence of illumination artifacts, such as specular reflections, which result from the use of a directional illumination source. It appears that there is a further need to solve the problem of illumination artifacts located on colonies in an image of a biological sample.
A further problem relates to the determination of the number of colonies, taking into account the colonies located in the periphery of the Petri dish. It appears that there is a need to improve the counting process of the colonies in an image of a biological sample.